Restek at ASMS 2024
2-6 June, Anaheim, CA, U.S.
Visit the ASMS 2024 website
Learn More About Featured Restek Products
- Pro EZLC Method Translator
- Raptor ARC 18 Columns
- Ultra IBD HPLC Columns
- Force Inert Biphenyl LC Columns
Questions about these presentations? Contact Restek Technical Service.
TECHNICAL POSTERS
Exploring Different HPLC Column Chemistries for Optimal Separation of 13 Bile Acids by LC-MS/MS
Wednesday, June 5
Jared Burkhart1, (presenter) Haley Berkland1, Paul Connolly1, Justin Steimling1, Katharina Schramm2, Tatiana Yuzyuk2
Restek Corporation, 2. ARUP Institute of Clinical and Experimental Pathology
Abstract
The analysis of bile acids in human plasma is an important diagnostic tool for detection of liver disease and as indicators of potentially harmful side effects of new drugs. There are two main types of bile acids based upon their functional groups: unconjugated bile acids and conjugated bile acids, primarily glycine- or taurine-bound. In this study, 13 bile acids were being analyzed by LC-MS/MS using a Raptor C18 50 x 2.1 mm, 1.8 μm column, but a matrix interference was identified to be coeluting with one of the bile acids standards and causing quantitation issues, so it was necessary to develop a new method on an alternative column chemistry.
Biphenyl, FluoroPhenyl, and ARC-18 stationary phases were tested on a 100 x 2.1 mm, 2.7 μm column dimension using water with 5 mM ammonium acetate as mobile phase A and 50:50 methanol:acetonitrile as mobile phase B. The column temperature was 50°C. Data was collected in ESI- mode.
The biphenyl stationary phase was able to partially resolve the matrix interference and showed selectivity for the taurine and glycine conjugated isomers, but limited selectivity for the unconjugated isomers. The fluorophenyl stationary phase was able to resolve the matrix interference but did not show selectivity for the three isomer sets. The ARC-18 stationary phase was able to resolve the matrix interference and showed selectivity for the three isomer sets. The gradient using the ARC-18 column was optimized to separate all 13 bile acids, including full resolution of the isomer sets and the matrix interference in a 9.5 minute cycle time.
Quantitation of bile acids in matrix is challenging for a number of reasons, such as structural similarities, varying polarity and stereochemistry, the presence of isomers, limited fragmentation of unconjugated bile acids in a mass spectrometer, high endogenous levels, and matrix effects caused by phospholipids or triglycerides. The developed LC-MS/MS method utilizing a Raptor ARC-18 column managed to overcome all of these challenges while also resolving a matrix interference that was preventing proper quantitation.
Method Development of PFAS Compounds Using a Virtual Method Development Tool
Wednesday, June 5
Melinda Urich, (presenter), Chris Nelson, Justin Steimling, Tim Yosca, Elena Gairloch
Restek Corporation
Abstract
Methods from organizations, including the International Organization for Standardization (ISO) and the Environmental Protection Agency (EPA), were selected for evaluation. Using the unique modeler tool, methods were developed virtually, tested experimentally, and retention times were compared to the results given by the modeler.
A library containing 58 PFAS compounds was used to virtually develop methods. Analytes were first selected, followed by column chemistry and dimension. Mobile phase, temperature, and gradient were modified to optimize isobaric separations and run time. Conditions were transferred to the instrumentation and data collected. To determine if results of the modeler were acceptable, retention times should not exceed more than 50% of a typical MRM window (±15 seconds) or no more than 10% of the runtime. Results show the modeler can develop and optimize separations quickly and accurately, offering the users the ability to increase throughput by saving time and cost.
Simultaneous Analysis of Ultrashort-Chain to Long-Chain (C1 to C10) and Alternative Per- and Polyfluorinated Substances in Human Plasma and Serum
Wednesday, June 5
Shun-Hsin Liang, (presenter), Justin Steimling
Restek Corporation
Abstract
The per- and polyfluoroalkyl substances (PFAS) targeted for analysis in blood may vary based on the specific concern in a given region or population. Some commonly analyzed PFAS range from short- to long-chain compounds (C4 – C10). The ultrashort-chain (USC) PFAS with carbon chain lengths of shorter than C4 have become a major concern due to their prevalence and high levels of occurrence in environmental aquatic systems. Several studies have indicated a rapid increase in environmental concentration of USC PFAS, raising the concern of elevated human exposure. The measurement of USC PFAS in blood can not only monitor the human exposure but also provide a tool for studying the potential risks associated with USC PFAS exposure.
Accuracy and precision test was conducted using fetal bovine serum fortified with C1 to C10 carboxylic acid and sulfonic acid PFAS. A 100 μL aliquot of serum sample was mixed with both extracted and nonextracted internal standards, along with 200 μL of methanol containing 1.5% formic acid. After centrifugation, 5 μL of supernatant was injected onto a polar-embedded, reversed-phase column (Ultra IBD, 100 x 2.1 mm) for chromatographic separation followed by MS/MS detection. Calibration standards were prepared in the range of 0.05 to 40 ppb in reverse osmosis water containing phosphate-buffered saline. The established method was then applied to measure PFAS in various human serum and plasma samples, including a NIST 1950 reference human plasma.
Simultaneous Determination of Alternaria Toxins, Ergot Alkaloid Epimers, and Other Major Mycotoxins in Various Food Matrices by LC-MS/MS.
Thursday, June 6
Diego A. Lopez, (presenter), Shun-Hsin Liang, Justin Steimling
Restek Corporation
Abstract
Various food commodities are vulnerable to different types of fungal pathogens and could be contaminated with differential classes of mycotoxins as a result. It is ideally to implement a generic method for simultaneous determination of multi-mycotoxins in different food matrices or agricultural products.
In this study, a simplified sample preparation procedure and a reliable LC-MS/MS analytical method was developed for comprehensive measurement of 37 regulated and emerging mycotoxins, including five Alternaria toxins, six major ergot alkaloids, and their corresponding epimers. Four different food matrices (baby wheat cereal, peanut, tomato puree, and blended flour) were chosen for method validation to demonstrate the applicability of this analytical method to a wide range of food types. Sample extraction was performed using a formic acid-acidified 80:20 acetonitrile:water solution followed by extract dry down and reconstitution in a 50:50 water:methanol solution for injection analysis on a Biphenyl LC column. Chromatographic analysis was performed using MS-friendly acidic mobile phases and completed with a short 11-minute cycling time for proper separation of ergot alkaloid epimers. Method accuracy and precision was evaluated by fortification of food samples at three different levels. Accurate quantification was achieved using matrix-matched calibration standards at the range of 0.4 to 400 μg/kg.
The recoveries of all mycotoxins (except citrinin) in fortified samples were from 70% to 120%, and the relative standard deviation was less than 20%. The established workflow was simple and fast for multi-mycotoxin determination with a unique benefit of simultaneous analysis of Alternaria toxins and ergot alkaloids. Furthermore, a novel inert Biphenyl LC column demonstrated the high degree of Non-Specific Binding (NSB) that occurs between the column’s stainless-steel hardware and certain mycotoxins.
The implementation of the inert column offers a robust and improved chromatographic performance as it mitigates the NSB for highly adsorptive analytes (e.g., fumonisins, aflatoxins, and tenuazonic acid) leading to better sensitivity and peak shapes without the need of mobile phase additives or sample passivation.